Bacterial cell division is a complex differentiation process achieved with remarkable fidelity. The division septum is formed at mid-cell so that cytosolic components are equi-partitioned into daughters. Selection of the correct division site in E. coli is accomplished by the cooperative action of a division inhibitor (MinC-MinD) and a topological specificity factor (Min E). We still do not understand their mechanism of action. A key to achieving such understanding will be determining the 3-dimensional structure of these proteins and the molecular details of their interactions. Specifically, it is planned to develop in vitro interaction assays to map regions of these proteins responsible for interactions with cognate partners, as well as the interaction of MinC with the cell division inhibitor DicB. Site directed mutagenesis as well as insertion and deletion mutants will be use to map regions of interaction. The phenotypic effects of these mutants will be examined and fluorescence microscopic examination will localize the cellular location of MinE point mutants lacking topological specificity. Limited proteolytic digestion will determine the core interacting domains of each protein. A combination of NMR and X-ray diffraction will then be used to determine structures of individual proteins, domains and protein complexes. The protein structures will be used for the structure based design of novel antibacterial agents.